ANTISENSE-MEDIATED INHIBITION OF PARATHYROID HORMONE-RELATED PEPTIDE PRODUCTION IN A KERATINOCYTE CELL-LINE IMPEDES DIFFERENTIATION

We have used antisense RNA technology to inhibit endogenous PTH-relate d peptide (PTHRP) production in an established human keratinocyte cell line, HPK1A, to assess the role of PTHRP as a modulator of cell diffe rentiation. Initially to determine the specificity of any alterations in cell function that might be observed, HPK1A cells and Rat-5 fibrobl asts (which do not synthesize PTHRP) were both infected with the same retrovirus (pYN) containing antisense PTHRP. In contrast. to antisense -infected HPK1A cells (HPK1A-AS), which show accelerated growth indice s when endogenous PTHRP production is blocked, antisense-infected Rat- 2 cells (Rat-2-AS) displayed no increase in cell proliferation. Conseq uently, this alteration in HPK1A cell function appeared to be specific to the inhibition of PTHRP production. In HPK1A-AS cells, no PTHRP tr anscript was observed in cytoplasmic RNA, and none was sequestered in a nuclear RNA preparation. Therefore, hybridization with the antisense strand appears to destabilize PTHRP mRNA, leading to rapid disappeara nce of the sense-antisense heteroduplex. We then examined the effect o f PTHRP inhibition on keratinocyte differentiation using three indices . PTHRP inhibition in HPK1A-AS cells resulted in reduced high mol wt k eratin production, as assessed by immunocytochemistry. Expression of m RNA encoding transglutaminase and involucrin was decreased in HPK1A-AS cells compared to that in control cells under conditions of high ambi ent calcium. Involucrin protein levels were also diminished in HPK1A-A S cells in parallel with the reduced levels of involucrin gene express ion. These data, therefore, show that interference with PTHRP producti on inhibits expression of maturation-specific keratinocyte indices and indicate that endogenous PTHRP acts to enhance differentiation in thi s keratinocyte model.